This invention relates to the field of liquid chromatography. More specifically, it relates to the design and construction of an improved detection system for use in a liquid chromatograph, particularly a high pressure liquid chromatograph.
One of the most pervasive problems in liquid chromatography, particularly high pressure liquid chromatography, is noise produced in the output of the detector by the operation of the high pressure pump. In particular, when a reciprocating pump is used, the base line of the detector output contains a noise spectrum, usually in the form of a series of spikes corresponding to the cycles of the pump. While this noise is low level noise (generally considerably smaller than 0.01 absorbance units) it does limit the sensitivity of the instrument.
Various attempts have been made to eliminate the noise in the output of the detector caused by the pump. In particular, more expensive constant flow pumps have been used, or pulse damping systems have been incorporated into the liquid line. With the exception of the use of constant pressure pumps, none of the approaches that have been tried have been overly successful, and all require complication and costly additional components.
The detector used in a liquid chromatography is most often a photometer comprising a radiation source, a flow cell and a radiation detector. The effluent from the chromatographic column passes through the flow cell, which is optically transparent, and changes in the chemical composition of the effluent are measured by monitoring the change in its optical density, using the radiation source and the radiation detector to measure the change in light transmission.
In such a system, the radiation from the radiation source heats up the optical cell, and the liquid in it, above ambient temperature. It has been found that the pulses observed in the output of the detector are due to changes in the index of refraction of the liquid in the optical cell. The index of refraction of a liquid is dependent on its temperature. When the effluent from a chromatographic column enters the flow cell, it does so at ambient temperature. As it flows through the cell, its temperature is raised by contact with the flow cell and it leaves the flow cell at an elevated temperature. This does not cause a major problem in a constant flow system, because some equilibrium always exists between the temperature of the liquid entering the flow cell and the temperature of the liquid leaving the flow cell, and the photometer measures the mean or average index of refraction.
The situation is quite different when a reciprocating pump or some other periodic pumping means is used. In such a system, there is a periodic pulse of liquid entering the flow cell at ambient temperature. There is, therefore, a difference in the equilibrium index of refraction during the pulse and the equilibrium index of refraction when the pulse stops. This difference appears in the output of the detector as noise, generally in the form of a spike.
There are a number of ways in which this particular problem can be solved. One way is to enclose the entire chromatographic system in an environment of controlled temperature, such as heat bath, which is maintained at a temperature equal to that of the flow cell. In this way, the liquid flowing through the chromatographic column and entering the flow cell would be at the same temperature as the liquid leaving the flow cell. Regardless of whether or not there were fluctuations in the flow of the fluid in the flow cell, the temperature of the liquid in the flow cell would be constant and the index of refraction of the liquid in the cell would also be constant.
Another way to achieve the same result is to monitor the temperature of the flow cell and the temperature of the effluent leaving the chromatographic column, and to actively control the temperature of the liquid leaving the chromatographic column until it is equal to the temperature of the flow cell.
Both of these techniques, however, are unduly complicated, and require expensive additional components. Applicant has found that the same result can be achieved by a simple, passive system.